4041bp: full length p53 tethered to full length p53 cDNA followed IRES expressed mCherry followed by SV40 polyA sequnece
crispr/cas9 insertion location
hg38 chr17:7,676,591
treatment
10uM Nutlin3a
chip antibody
DO-1 (BD Biosciences, BD554293)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Six hours after the addition of 10 mM Nutlin-3a or 0.1% DMSO, cells (~60 x 10^6 cells) were fixed with 1% formaldehyde for 10 min at 25oC. Glycine (0.125 M) was then added for 5 min. Cells were scraped into conical tubes and washed with ice cold PBS. Nuclei were isolated by resuspending the cells in NRO buffer (80 ml/million cells; 10 mM Tris-HCl [pH 8], 4 mM MgCl2, 10 mM NaCl, 0.5% [vol/vol] NP-40 and the protease inhibitor cocktail: 0.25 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium metabisulfite, 0.012 TIU/mL aprotinin, 1 mM benzamidine and 1 mM dithiothreitol (DTT)), followed by a 5 min incubation on ice, a low speed spin, and then a final wash with NRO buffer. The isolated nuclei were prepared for shearing based on the Covaris truChIP Chromatin Shearing Kit (Covaris: PN 520237) and sheared for 11 min on Covaris M220 Focused-ultrasonicator. Samples were spun at high speeds then a small aliquot for input was removed while the rest of the supernatant containing the sheared chromatin was transferred to prepared Protein G Dynabeads (Invitrogen: 10003D). 25mL of Protein G Dynabeads beads per pull down were washed 3 times in block solution (PBS, 0.5% BSA) before being resuspended in block solution with 6 mg of DO1 p53 antibody (BD Biosciences: BD554293) per pull down. The beads and antibody were allowed to nutate for 10 min at 25oC before nutating at 4oC for 4 hours. Beads were then washed in block solution once and IP buffer once (15 mM Tris-HCl [pH 8], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and the protease inhibitor cocktail). Beads were then resuspended in IP buffer and the sheared chromatin was added. The sample was then allowed to nutate at 4oC for at least 12 hours. The bound chromatin was then washed 3 times with IP buffer, 3 times with RIPA buffer (20 mM Tris-HCl [pH 8], 500 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 0.1% SDS), 2 times with LiCl buffer (20 mM Tris-HCl [pH 8], 500 mM LiCl, 1 mM EDTA, 1% sodium deoxycholate and 1% NP-40) and 2 times with TE Salt buffer (10 mM Tris-HCl [pH 8], 50 mM NaCl, 1 mM EDTA). The sample was then eluted with PK buffer (10 mM Tris-HCl [pH 8], 1 mM EDTA and 1% SDS) containing 100 mg of Proteinase K (New England BioLabs: P81075) per reaction by incubating at 50oC shaking for 1-hour then at 65oC for 1-hour. The supernatant containing the eluted DNA was transferred to a new tube for reverse crosslinking. The input was thawed and brought up to sample volume with PK buffer containing 100 mg of Proteinase K per reaction. All samples and inputs were incubated at 65oC for at least 12 hours. DNA was then purified by phenol chloroform extraction using Light-5PRIME Phase Lock Tubes (Quanta Bio: 2302820) based on the manufacturer's instructions. Libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems: KK8502) and sequenced on Illumina NextSeq V2 high output 75-cycle.